Data and code from Durrieu et al. (IScience, 2022). Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources

Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources

Several assays exist to measure nuclear transport rates, but not at the single-cell level. Here, we developed a fluorescent recovery after
photobleaching (FRAP)- based method to determine nuclear import and export rates independently, in
individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential
FRAPs on the same cell and optimization to an elaborate model that accounts for both the biological and the experimental aspects. We found large cell-to-cell variation in transport rates within isogenic yeast
populations. Our data suggest that a main determinant of this heterogeneity may be variability in the number
of nuclear pore complexes (NPCs). For passive transport, this component explained most of the variability.
Actively transported proteins were influenced by variability in additional components, including general
factors such as the Ran-GTP gradient as well as specific regulators of the export rate. By considering
mother-daughter pairs, we showed that mitotic segregation of the transport machinery is too noisy to control
cellular inheritance. Finally, we studied mother-daughter cell asymmetry in the localization of the
transcription factor Ace2, which is specifically concentrated in daughter cell nuclei. We found that this
asymmetry is the outcome of a higher ratio of import rate to export rate in daughters.

Uploaded here:
Code
- the NuclearFRAP R package, for the fitting of FRAP experiments in single cells.
- Matlab scripts for simulation of data from FRAP experiments
- R scripts for analysis of the data (internal scripts used to create the plots from the paper, comments are mostly in spanish)

Data
- Raw data of train of FRAP experiments in yeast. Performed on the proteins YFP, Fus3-YFP, YFP-Ace2 and YFP-Ace2 G128E.
- Results of fitting the experimental data of train of FRAP experiments in yeast. Output from the NuclearFRAP package.
- Simulated train of FRAP experiments raw data (for different signal/noise levels)
- Results of fitting the simulated train of FRAP data
- Raw measurements of the abundance of nuclear pore complex proteins. Result of the quantification of confocal images of the proteins Mpl1-GFP, Mpl2-GFP, Prm3-GFP and Nup49-GFP.
- Raw data from the train of FRAPs experiments on Ace2, in cells Crm1* treated with LMB.

Methods and protocols
- Protocols from the lab for performing and analyzing the train of FRAPs and NPC quantification. In spanish.

The microscopy images from the train of FRAPs experiments are available upon request. For more data or any questions please write to: [e-mail]

对同源酵母种群中核质运输速率的细胞间差异进行表征及其来源识别

目前存在多种方法用于测量核质运输速率，但均未达到单细胞水平。在本研究中，我们开发了一种基于荧光漂白后恢复（FRAP）的方法，以独立测定单个活细胞中的核输入和输出速率。为了克服单细胞测量的固有噪声，我们对同一细胞进行了连续的FRAP实验，并优化了详尽模型，该模型同时考虑了生物学和实验方面的因素。我们发现，在相同的同源酵母种群中，核质运输速率存在显著的细胞间差异。我们的数据表明，这种异质性的主要决定因素可能是核孔复合体（NPCs）数量的可变性。对于被动运输，这一因素解释了大部分的变异性。而主动运输的蛋白质则受到其他成分变异性影响，包括诸如Ran-GTP梯度等一般因素以及特定调节输出速率的因子。通过考虑母细胞-子细胞对，我们发现运输机器的减数分裂分离过于嘈杂，无法控制细胞遗传。最后，我们研究了转录因子Ace2在母细胞-子细胞中的定位不对称性，Ace2在子细胞核中特异性富集。我们发现，这种不对称性是子细胞中输入速率与输出速率比率更高的结果。

所上传材料包括：

- 核质FRAP R包，用于单细胞FRAP实验的拟合。

- 用于模拟FRAP实验数据的Matlab脚本。

- 分析数据的R脚本（用于从论文中创建图表的内部脚本，注释大多为西班牙语）。

- 酵母中FRAP实验序列的原始数据。

- 酵母FRAP实验序列实验数据的拟合结果。

- 不同信号/噪声水平的模拟FRAP实验原始数据。

- 模拟FRAP数据拟合结果。

- 核孔复合体蛋白质丰度的原始测量数据。

- 蛋白质Mpl1-GFP、Mpl2-GFP、Prm3-GFP和Nup49-GFP共聚焦图像定量结果。

- 在细胞Crm1*用LMB处理的细胞中进行的Ace2 FRAP实验的原始数据。

方法与方案：

- 实验室进行和分析FRAP序列及NPC计数的方案。方案大多为西班牙语。

显微镜图像可应要求提供。如需更多数据或有任何疑问，请联系：[e-mail]